Purine Salvage in Relapsing Fever Spirochetes[unreadable] Spirochetes in the genus Borrelia are comprised of two phylogenetic branches for which one group is exemplified by the relapsing fever spirochetes and the other group is represented by the Lyme disease spirochetes. A striking difference in the pathogenicity of these bacteria is the ability of the relapsing fever spirochetes to achieve much higher cell densities in blood than does the Lyme disease spirochete Borrelia burgdorferi. The mechanisms underlying this difference in levels of spirochetemia are a primary focus of our research. Genome sequencing projects in our laboratory with two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed the presence of genes involved in purine and glycerol metabolism that are absent in the Lyme disease spirochetes. We hypothesized that the more complete pathways for the utilization of these important metabolites by the relapsing fever spirochetes contribute to their growth to higher cell densities in the blood. To define the importance of the B. hermsii gene products for the acquisition of the purine hypoxanthine from the hosts blood, novel genetic constructs were developed to inactivate genes in this purine salvage pathway. Methods were also developed to reintroduce the wild-type gene into the inactivation mutants to restore the wild-type phenotype. Preliminary infection studies in mice with B. hermsii containing mutations in the operon for the salvage of hypoxanthine showed reduced levels of spirochetemias and no detectable relapse compared to wild-type spirochetes. However, we were concerned that microscopic quantification of spirochetes in the blood might not be sensitive enough to elucidate differences between the mutant and wild-type spirochetes. Therefore, efforts were made to develop a quantitative PCR to better assess the possible reduction in the number of spirochetes in which this metabolic pathway has been disrupted. A quantitative PCR (QPCR) was developed to count B. hermsii in the blood of infected mice, and these results were compared to estimates based on microscopy. Three mice were infected via needle inoculation with wild-type B. hermsii and bled each day for 12 consecutive days. The estimated numbers of spirochetes per ml of mouse blood were very similar with both techniques, however, the QPCR was slightly more sensitive, allowed for more replicates and statistical analysis, and was much faster. Counting spirochetes in the 36 samples of blood using stained smears and microscopy took about 30 hours, whereas, all the samples with 6 replicates each were completed in approximately one afternoon using QPCR. Thus, we are now prepared to examine further the effect on the genetic inactivation of one arm of the purine salvage pathway in B. hermsii in relation to the spirochete's ability to grow in the host's blood. [unreadable] [unreadable] Birds as potential hosts for relapsing fever ticks [unreadable] Previously, we identified two genomic groups in B. hermsii via multilocus sequence typing. Other investigators recently typed a small number of relapsing fever spirochetes based on the intergenic spacer (IGS) region of noncoding DNA located between the 16S rRNA and ileT tRNA genes. Therefore, we examined the IGS locus in 37 isolates of B. hermsii and compared the results to those obtained by multilocus sequence typing (Schwan et al., 2007 in bibliography). The phylogram based on the IGS sequences separated the 37 isolates of B. hermsii into GGI and GGII as defined previously. The presence of multiple genotypes of B. hermsii separated over large geographical distances, suggested that birds might play a role in dispersing these spirochetes in nature. For this to occur, birds must be suitable hosts for O. hermsi. Therefore, we evaluated the competence of birds as hosts for both the ticks and spirochetes in the laboratory. Chickens and bobwhite quail were used to feed ticks. Larvae, nymphs and adults all fed quickly on the birds and the ticks survived with little mortality. Females laid viable eggs that produced larvae, which survived 7 months until they fed on mice. Larvae that fed on chickens molted to first nymphs and they then fed on quail or mice up to 9 months later. These results demonstrated that birds were suitable hosts for all stages of O. hermsi.[unreadable] [unreadable] Factor H binding protein gene in Borrelia hermsii [unreadable] Recently, Hovis and colleagues (2006, Infection and Immunity 74:4519-4529) examined isolates of B. hermsii to characterize the gene encoding the spirochete protein that binds factor H and the factor H-like 1 protein. Two sequence groups of the factor H binding gene were identified, fhbA1 and fhbA2, and the numerical designations were chosen to correspond to the two genomic groups of B. hermsii we defined by multilocus DNA sequence typing. By our analysis, five chromosomal loci, including 16S rRNA, flaB, gyrB, glpQ, and the Intergenic Spacer (IGS) region, contained sequences that placed all isolates of spirochetes in either Genomic Group I (GGI) or Genomic Group II (GGII). In contrast, Hovis and colleagues found that 6 of 14 GGI isolates had fhbA sequences representative of GGII isolates. The authors suggested that the discrepancy might have arisen through the lateral transfer of a 30 kb fhbA-containing prophage between spirochetes in the two genomic groups. The authors also reported that three of 24 isolates were mixed infections because both fhbA1 and fhbA2 were found by PCR analysis using primers specific for each gene type. Given our interest in the mechanisms of pathogenicity and genetic diversity of B. hermsii, and the possible lateral transfer of fhbA between spirochetes, we performed an analysis to determine the fhbA genotype of our B. hermsii isolates. Since we had provided the isolates used by the other investigators for their analysis of the fhbA locus, the material used in both studies was identical but expanded in our study.[unreadable] Our results conflict with data and conclusions of Hovis et al. First and most important is our finding of total congruence between the B. hermsii fhbA type and genomic group. All isolates with fhbA1 were in GGI while all isolates with fhbA2 were in GGII. Therefore, we found no evidence for the lateral transfer of this locus from spirochetes in one genomic group to the other. Second, we found only 3 fhbA alleles among the 34 isolates, while Hovis et al. reported 7 alleles in 10 of the isolates. We sequenced amplicons produced with primers that flanked the ORF. Hovis et al. sequenced amplicons produced with primers with sequences within the ORF that were designed from the fhbA2 sequence of B. hermsii YOR, a GGII isolate. By using primers with sequences unique to fhbA2 to amplify fhbA1 from GGI isolates, erroneous bases were apparently incorporated into the fhbA1 amplicons. Third, our PCR analysis using the fhbA1 and fhbA2 type-specific primers did not support the claim by Hovis et al. that some of the isolates contained both fhbA types, as none of the isolates produced amplicons with both primer sets.[unreadable] We do not know the basis for the discrepancy between our results and those of Hovis et al., but they cannot be due to a different source of material as the same isolates were included for both studies. One possible and likely explanation is that the other investigators cross-contaminated the isolates or DNA after they received the samples from us, resulting in mixed PCR products and DNA sequencing of non-representative cloned fragments. Regardless, the fhbA locus defined the genomic group for each isolate of B. hermsii, as do several other loci. The significance of factor H binding by B. hermsii toward the pathogenesis of relapsing fever remains to be determined.